| Assay Method Information | |
| | Inhibition of KIF18A Microtubule-Dependent ATPase Activity |
| Description: | Table 9: Test compounds were plated in a 3× dilution scheme in a 384-well plate. Assay buffer: 80 mM PIPES (pH 6.9), 1 mM MgCl2, 75 mM KCl, 1 mM EGTA, 1 mM DTT, 0.01% BSA, 0.005% Tween-20, 1 μM Taxol in H2O. To 50 nL of compound in DMSO was added 2.5 μL of enzyme mix [4 nM hKIF18A (1-374) in assay buffer]. After incubation at room temperature for 30 min, 2.5 μL of microtubule mix was added [0.2 mg/mL pre-formed microtubules, 2.0 mM ATP in assay buffer], the plate was centrifuged for 30 s and then incubated at 28° C. for 60 min. 5 μL of Promega ADP-Glo Max R1 was added, the plate was centrifuged for 30 s, and the mixture incubated for 4 h at room temperature. 10 μL of Promega ADP-Glo Max R2 was added, the plate centrifuged for 30 s, and incubated for 60 min at room temperature. Luminescence was measured with an Envision plate reader, and % Inhibition was calculated for each well as: ([max−min]−[test−min])/[max−min]. IC50 values were calculated from concentration vs. % Inhibition data via a four-parameter variable slope model. |
| Affinity data for this assay | |
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